Formulation of pharmaceutical proteins presents significant challenges. Proteins possess multiple functional groups in addition to three-dimensional structure; degradation therefore proceeds via both chemical (modifications involving bond formation or cleavage) and physical (denaturation, aggregation, adsorption, precipitation) pathways. Since each protein embodies a unique combination of amino acid sequence, isoelectric point, and other determinants, its response to changes in solution conditions is unpredictable, and must be determined on a case-by-case basis. Attempts to prevent one form of degradation often increase the rate of another.
Degradation of proteins can be greatly reduced or avoided by lyophilization. However, lyophilization is time consuming and costly, and can cause protein denaturation and aggregation if appropriate excipients are not included. As with solution formulation, stabilization of lyophilized proteins must be dealt with on an individual basis. Sodium chloride can be used to maintain stability during purification and storage by reducing aggregation and precipitation. However, sodium chloride is a problematic excipient in lyophilized formulations because it lowers glass transition temperature, thereby necessitating low primary temperatures and long cycle times. Jiang et al, US 20060002918 A1 disclose methods for stabilizing lyophilized thrombin preparations by formulating the thrombin with sucrose, mannitol, sodium chloride, and a surfactant or high molecular weight polyethylene glycol. However, stabilization of a protein in the lyophilized state does not ensure stability upon reconstitution, and protein solutions must often be discarded if not used promptly after reconstitution. In addition, the in-use handling of a lyophilized drug product is often limited by sterility considerations because the container-closure integrity is compromised during reconstitution. As such, the in-use handling time could be extended if a drug product formulation was stable in the presence of a preservative.
In view of the often high cost of protein therapeutics, means of enhancing the stability of these proteins in solution are needed. In the case of thrombin, various excipients have been proposed for stabilizing the protein. For example, Silbering et al., U.S. Pat. No. 4,696,812 disclose the use of low levels of saline and glycerol to reduce denaturation of thrombin in solution. Nishimaki et al., U.S. Pat. No. 5,130,244 disclose an aqueous thrombin solution containing a sugar and an amino acid as stabilizers.
Despite these advances, there remains a need in the art for preparations of thrombin that can be stored for extended periods of time in liquid form.